Development and validation of a conditional localization domain to control trafficking of secretory proteins in plasmodium falciparum

Plasmodium falciparum;apicoplast;Cell Biology

Roberts, AleahMcMacken, Roger ;

Johns Hopkins University

关键词: Plasmodium falciparum;    apicoplast;    Cell Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/44710/ROBERTS-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The apicoplast is an essential organelle of P. falciparum. Currently, there are few molecular tools to identify and validate specific essential apicoplast proteins. We have therefore developed a molecular tool to probe the function of apicoplast-targeted proteins.Nuclear-encoded proteins that are targeted to the apicoplast contain a transit peptide sequence that is essential for sorting proteins from the endoplasmic reticulum, into vesicles that fuse with the apicoplast outer membrane. Transit peptides have a unique requirement that they are unstructured during apicoplast import; furthermore, formation of structure in the transit peptide region blocks import. The goal of my thesis was to use this feature of transit peptides to design a conditional localization domain (CLD) tag that can control the localization of an apicoplast targeted protein. The CLD replaces the natural transit peptide of the target protein and traffics to the apicoplast under permissive conditions. An interacting ligand is added to the cell to bind the domain and cause the CLD to become secreted.We tested several proteins as potential CLDs and analyzed their ability to control trafficking of a fluorescent cargo protein using microscopy. Chapters 2 and 3 describe our efforts to design a suitable CLD from Dihydrofolate Reductase (DHFR) enzymes and the FK-506 Binding Protein (FKBP). The candidate domains we designed from DHFR were not suitable CLDs because they did not traffic to the apicoplast. Our initial design of a CLD from FKBP was also not a suitable CLD because it was too unstable to respond to the interacting ligand, although it was able to traffic to the apicoplast. We modified the original CLD designed from FKBP to generate three successful domains (CLD1, 2, and 3) that can be used to control the trafficking of apicoplast-targeted proteins. In Chapter 4 we analyzed the trafficking dynamics of CLD1, 2, and 3 in detail. Results show that CLD2 and CLD3iitraffic to the apicoplast more efficiently than CLD1; we found that CLD1’s leaky apicoplast trafficking is caused by a higher protein stability compared to CLD2 or CLD3.To validate this system, we tested whether CLD1 or CLD2 could conditionally localize a parasite biotin ligase called Holocarboxylase Synthetase 1 (HCS1) without interfering with the function of the enzyme. HCS1 biotinylates the acetyl-CoA carboxylase, a protein that is only biotinylated in the apicoplast of liver stage parasites. In parasite lines that express CLD1-HCS1 or CLD2-HCS1, we were able to control protein biotinylation in the apicoplast in a ligand-dependent manner, demonstrating the full functionality of the CLD tool.